The overall objective of research at the Buck Institute for Age Research is to understand aging and its associated diseases in order to extend the healthy years of human life. Research at the Buck Institute is reaching the stage at which techniques perfected for isolated cells can now be applied to more complex acute slice and in vivo models. This requires an appropriate microscope facility and this shared instrumentation proposal seeks funding for the purchase of a Leica TCS SP5 MP upright fluorescence microscopy system for two-photon and confocal imaging. The instrument would be used by projects that are examining specific key issues in human health related to aging, neurodegenerative disease, stroke, cancer and stem cell therapies. The existing live specimen confocal instrumentation comprises a Zeiss LSM510 with a Coherent Chameleon XR modelocked Ti:Sapphire (two-photon) laser. Since this is mounted on an Axiovert 200 which is an inverted microscopic system, with lenses and internal, descanned detectors with filter-wheel based spectral selection optimized for cell culture and histological work, it is inappropriate for thick slice and whole animal studies. The requested Leica TCS SP5 MP will include an upright microscope, deep tissue optimized dipping lenses and reflected and transmitted light non-descanned detectors for two-photon microscopy. Additionally, a 442 nm and other standard visible lasers, in combination with three internal detector spectral imaging will facilitate versatle single photon, confocal microscopic use. The system will be integrated with an existing Coherent Chameleon XR two-photon laser, while the Zeiss LSM510 will be re-equipped with a 405nm violet laser better suited for the thin sample UV excitation. Funding for the latter will be obtained from a budget separate from this application. The new Leica instrument will immediately enhance the work of multiple research groups at the Buck Institute who have significant needs in deep-tissue fluorescence imaging or spectral confocal microscopy. The most critical features of this new instrumentation will be the capability to (1) conduct deep-tissu time lapse imaging of physiological parameters and morphology using fluorescent probes and markers at depths of up to 600?m in vivo or in ex vivo living samples, (2) allow three dimensional reconstruction of fixed samples at depths of up to 1.5 mm, and (3) facilitate spectral scanning and simultaneous spectral separation of up to three individual fluorophores in a single sample, features that we currently lack. Specific projects will investigate oxidative stress, reactive oxygen species, organelle transport and energetics, protein aggregation and dendritic spine structure in neurodegenerative diseases; migration of transplanted neuronal precursor cells; genomic field defects in preneoplastic breast cancer, an in vivo fly model of kidney disease, and pharmacokinetic imaging of a compound that slows aging in a worm model of human aging. PUBLIC HEALTH RELEVANCE: Ten independent research groups at the Buck Institute will use this new technology to improve and expand their ongoing studies into the mechanisms and basic biology of human aging and into diseases whose incidence increases significantly with age, including Huntington's, Alzheimer's and Parkinson's diseases, breast cancer, and stroke. The overarching aim is to reduce the incidence and severity of these diseases and extend the healthy years of life. The relevance of this new instrumentation to public health is significant in that the benefits to society will ultimately include both increased quality of later life, and significant decreases in healthcare costs.